It now seems clear that platelet production is controlled by a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin). The studies necessary to determine the chemical characteristics, site of production, mechanism(s) of action, and mechanism(s) that sense the need for TSF release and action have not been performed because of the lack of suitable assay techniques and availability of a stable, purified source of TSF. Research described in this proposal will provide new assay procedures and a suitable supply of TSF for further studies. The objectives of this proposal are to: (1) develop bio- and immunoassays for TSF; (2) produce TSF in vitro and make comparisons to TSF produced from different sources; (3) purify TSF; (4) produce antibodies to TSF; (5) establish the site of production of TSF; (6) clarify the mechanism(s) that cause release of TSF in vivo; (7) establish cell lines that produce TSF; (8) determine changes in ploidy of megakaryocytes after TSF treatment; (9) investigate megakaryocytopoiesis both in vivo and in vitro; and (10) test TSF in sera of patients with platelet production disorders. These objectives will be pursued by utilizing a variety of physical, chemical, and immunological methods: (1) sera from thrombocytopenic animals will be used as a source of TSF; (2) both human embryonic kidney cell cultures and specific tumor cell lines will be utilized for in vitro production; (3) anti-TSF sera will be raised in animals and by use of monoclonal antibody technology; (4) purification of TSF from various sources will be made by high performance liquid chromatography and immunologic techniques; (5) new assay techniques and organ removal followed by dialysis will be used for determination of site(s) of production of TSF; (6) measurement of megakaryocytopoiesis after injection of potent sources of TSF will be investigated; (7) in vitro megakaryocytic colonies will be produced from murine and human bone marrow and K562 cells after stimulation of TSF; and (8) splenectomy, hypoxia, and 89Sr will be used for the study of TSF and erythropoietin interactions. These studies will provide the necessary information to predict the mode of action of thrombopoietin, site of production, and mechanism(s) that sense its need in animals. In addition, the development of assay procedures for TSF, production of monoclonal antibodies and purification of the hormone will make possible studies of TSF action in patients with platelet production disorders.